WO2017126626A1 - Antitumor agent - Google Patents

Antitumor agent Download PDF

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WO2017126626A1
WO2017126626A1 PCT/JP2017/001803 JP2017001803W WO2017126626A1 WO 2017126626 A1 WO2017126626 A1 WO 2017126626A1 JP 2017001803 W JP2017001803 W JP 2017001803W WO 2017126626 A1 WO2017126626 A1 WO 2017126626A1
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Prior art keywords
ferrichrome
group
administered
cancer cell
antitumor agent
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PCT/JP2017/001803
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French (fr)
Japanese (ja)
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幹浩 藤谷
弘晃 小西
健太郎 盛一
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国立大学法人旭川医科大学
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Priority to EP17741503.1A priority Critical patent/EP3400952B1/en
Priority to JP2017562903A priority patent/JP6830255B2/en
Priority to US16/071,610 priority patent/US10576126B2/en
Publication of WO2017126626A1 publication Critical patent/WO2017126626A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/12—Cyclic peptides, e.g. bacitracins; Polymyxins; Gramicidins S, C; Tyrocidins A, B or C
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/555—Heterocyclic compounds containing heavy metals, e.g. hemin, hematin, melarsoprol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K33/00—Medicinal preparations containing inorganic active ingredients
    • A61K33/24—Heavy metals; Compounds thereof
    • A61K33/26—Iron; Compounds thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • the present invention relates to an antitumor agent, in particular, an antitumor agent having a low risk of side effects and excellent antitumor effect, and use thereof.
  • Colorectal cancer that develops in the large intestine (cecum, colon, rectum) is ranked third in men and first in women as a cancer type with a high mortality rate in Japan in 2014.
  • the object of the present invention is to provide a safe and highly effective antitumor agent.
  • the present inventors have found a substance having high antitumor activity in the culture supernatant of Lactobacillus bacteria, and have completed the following inventions.
  • An antitumor agent comprising a compound represented by the following formula (1) or a complex thereof as an active ingredient.
  • R 1 represents a hydrogen atom or a hydroxymethyl group
  • R 2 represents a hydrogen atom, a methyl group or a hydroxymethyl group
  • R 3 , R 4 and R 5 each independently represents a methyl group, N 5 - (trans-5-hydroxy-3-methylpent-2-enoyl) group, N 5 - (cis-5-hydroxy-3-methylpent-2-enoyl) group or N 5 - (trans-4-carboxy -3 -Methylpent-2-enoyl) group.
  • R 1 and R 2 are hydrogen atoms
  • R 3 to R 5 are all methyl groups.
  • the anti-tumor activity equivalent to or better than 5-fluorouracil or cisplatin used in clinical practice as an anti-tumor agent while being effective and safe without causing adverse events such as hepatotoxicity. It is possible to provide a high antitumor agent.
  • Lactobacillus GG ATCC 53103, a L. lactobacillus bacterium. casei ATCC 334, L.C. Coryniformis ATCC 25600 and L. 2 is a graph showing the antitumor activity of each culture supernatant of fermentis ATCC 23271 against colon cancer cell lines.
  • Panel A shows anti-tumor activity against colon cancer cell line Caco2 / bbe
  • Panel B shows colon cancer cell line SKCO1
  • Panel C shows anti-tumor activity against colon cancer cell line SW620.
  • L. 2 is a HPLC chromatography chart of a compound showing antitumor activity purified from a culture supernatant of casei ATCC334.
  • Panel A is a fraction showing the purified anti-tumor activity
  • Panel B is a chart showing the results of mass spectrometry of commercially available ferrichrome. It is a graph which shows the dose dependence of the antitumor activity of ferrichrome with respect to colon cancer cell line Caco2 / bbe (panel A) and colon cancer cell line SW620 (panel B).
  • FIG. 6 is a graph showing the effect of ferrichrome on rat intestinal epithelial cells IEC-18 (panel A) and mouse primary intestinal epithelial cells (panel B). It is a photograph showing the growth of a tumor mass when ferrichrome is administered to a nude mouse transplanted with a colon cancer cell line SW620.
  • FIG. 6 is a graph comparing the antitumor effects of ferrichrome (panel A), 5-fluorouracil (panel B) and cisplatin (panel C) on colon cancer cell line SW620.
  • FIG. 6 is a graph comparing the effects of ferrichrome (panel A), 5-fluorouracil (panel B) and cisplatin (panel C) on rat intestinal epithelial cells IEC-18.
  • FIG. 1 It is a photograph showing the results of measuring the expression of phospho-Akt, JNK, ERK, p38MAPK and GSK3 ⁇ in a colon cancer cell line SW620 treated with ferrichrome by Western blotting. It is a graph which shows the change of anti-tumor activity when SP600125 which is an inhibitor of JNK pathway is added to colon cancer cell line SW620 with ferrichrome. It is a graph which shows the dose dependence of the antitumor activity of ferrichrome with respect to pancreatic cancer cell lines bxpc3 (panel A) and suite2 (panel B). The vertical axis represents cell density (OD at 510 nm), and the horizontal axis represents culture time after addition of ferrichrome.
  • FIG. 1 The vertical axis represents cell density (OD at 510 nm), and the horizontal axis represents culture time after addition of ferrichrome.
  • FIG. 6 is a graph showing the dose dependence of antitumor activity of ferrichrome against gastric cancer cell lines MKN45 (panel A) and SH-10-TC (panel B).
  • the vertical axis represents cell density (OD at 510 nm), and the horizontal axis represents culture time after addition of ferrichrome.
  • the vertical axis represents cell density (OD at 510 nm), and the horizontal axis represents culture time after addition of ferrichrome. It is a photograph which shows the growth of the tumor mass when ferrichrome is administered to the nude mouse which transplanted gastric cancer cell line MKN45.
  • the 1st aspect of this invention is related with the antitumor agent which uses the compound or its complex shown by following formula (1) as an active ingredient.
  • R 1 represents a hydrogen atom or a hydroxymethyl group
  • R 2 represents a hydrogen atom, a methyl group or a hydroxymethyl group
  • R 3 , R 4 and R 5 each independently represents a methyl group, N 5 - (trans-5-hydroxy-3-methylpent-2-enoyl) group, N 5 - (cis-5-hydroxy-3-methylpent-2-enoyl) group or N 5 - (trans-4-carboxy-3 Methylpent-2-enoyl) group.
  • the compound represented by the formula (1) is a kind of siderophore (a chelate compound derived from a microorganism that forms a stable complex with an iron ion), for example, as described in Patent Document 1 (Japanese Patent Laid-Open No. 2009-28030). It is a kind of protein. A compound in which R 1 and R 2 in the formula (1) are hydrogen atoms and R 3 to R 5 are all methyl groups is generally called ferrichrome. In Patent Document 1, the compound represented by the formula (1) is reported as a siderophore produced by Aspergillus oryzae, which is a type of filamentous fungus.
  • the inventors of the present invention have developed a culture supernatant of L. casei ATCC 334, which is a type of probiotics registered and stored in the American Type Culture Collection (ATCC), for colon cancer, pancreatic cancer, It showed strong antitumor activity against the growth of various cancer cells such as gastric cancer and esophageal cancer, and confirmed that such activity was brought about by ferrichrome.
  • ATCC American Type Culture Collection
  • more than 260 types of siderophores have been reported (SideroforeBase, http://bertrandsamuel.free.fr/siderophore_base/index.php), but it is novel that ferrichrome has antitumor activity. It is knowledge.
  • the metal ion of the compound shown by Formula (1) especially the complex with an iron ion is also included in the compound utilized in this invention.
  • the compound represented by the formula (1) can be produced by culturing Aspergillus oryzae strain 3129-7 (FERM P-20961) under the culture conditions described in Patent Document 1.
  • ferrichrome is L. casei, especially L. Casei ATCC334 can also be produced by culturing in an appropriate medium.
  • L.M. Casei ATCC334 is obtained by culturing in a liquid medium suitable for culturing Lactobacillus bacteria such as Man-Rogosa-Sharp (MRS) broth (Difco), Minimal Essential Media (Thermo Fisher Scientific) From the culture supernatant, it can be produced by appropriately combining gel filtration, reverse phase chromatography, ion exchange chromatography and the like.
  • MRS Man-Rogosa-Sharp
  • Minimal Essential Media Minimal Essential Media
  • ferrichrome can also be chemically synthesized according to the method described by Isova (Bulletin of the Chemical Society of Japan 47 (1), 215-220, 1974).
  • the compound of the formula (1) may be used as an antitumor agent as it is, and further, a pharmaceutically acceptable buffer, stabilizer, preservative, excipient and other components and / or others. It may be used in the form of a pharmaceutical composition containing the active ingredients. Such a pharmaceutical composition is the second aspect of the present invention.
  • Pharmaceutically acceptable ingredients are well known to those skilled in the art, and those skilled in the art can use the ingredients from the ingredients described in the 16th revised Japanese Pharmacopoeia and other standards within the scope of their normal performance. Can be selected and used as appropriate.
  • the form of the pharmaceutical composition containing the antitumor agent of the present invention is arbitrary, and it may be a parenteral preparation such as an injection or infusion, or an oral administration agent optionally coated with an appropriate coating.
  • a parenteral preparation such as an injection or infusion
  • an oral administration agent optionally coated with an appropriate coating.
  • carriers that can be used for parenteral preparations include aqueous carriers such as physiological saline, isotonic solutions containing glucose, D-sorbitol and the like.
  • the administration method of the antitumor agent of the present invention or the pharmaceutical composition containing the same is not particularly limited. However, in the case of a parenteral preparation, for example, intravascular administration (preferably intravenous administration), intraperitoneal administration, or intestinal administration. And subcutaneous administration.
  • the therapeutic agent of the present invention is administered to a living body by intravenous administration or oral administration.
  • the antitumor agent of the present invention or a pharmaceutical composition containing the same may be used in combination with other drugs useful for the treatment of tumors.
  • the dose of the antitumor agent of the present invention or the pharmaceutical composition containing the same is appropriately selected according to the usage, the age of the patient, the form of the disease, other conditions, etc., but usually 10 ⁇ g per kg body weight for an adult. It is ⁇ 2000 ⁇ g, preferably 50 ⁇ g to 1000 ⁇ g, more preferably 100 ⁇ g to 500 ⁇ g, and this can be administered once or several times a day or intermittently.
  • the antitumor agent of the present invention or the pharmaceutical composition containing the same can be used for the prevention and / or treatment of tumors, particularly digestive organ cancers. Therefore, the third aspect of the present invention is the above-mentioned It can be said that the present invention also provides a method for the prevention and / or treatment of tumors, particularly gastrointestinal cancers, using a compound of formula (1), particularly ferrichrome.
  • ⁇ Cell lines, microorganisms and their culture> In colon cancer cell lines Caco2 / bbe (ATCC), SKCO-1 (ATCC) and SW620 (ATCC), pancreatic cancer cell lines bxpc3 (ATCC) and Suite2 (Human Science Resource Bank), in gastric cancer cell lines MKN45 (National Research Institute for Healthcare and Nutrition, JCRB Cell Bank) and SH-10-TC (Medical Cell Resource Center / Cell Bank), esophageal cancer cell line OE33 (DS Pharma), and rat Intestinal epithelial cell strain IEC-18 (ATCC) was used for evaluation.
  • Caco2 / bbe ATCC
  • SKCO-1 ATCC
  • SW620 ATCC
  • pancreatic cancer cell lines bxpc3 ATCC
  • Suite2 Human Science Resource Bank
  • gastric cancer cell lines MKN45 National Research Institute for Healthcare and Nutrition, JCRB Cell Bank
  • SH-10-TC Medical Cell Resource Center / Cell Bank
  • OE33 esophageal cancer
  • Caco2 / bbe, SKCO1 and Suite2 are in DMEM, SW620, bxpc3, MKN-45, SH-10-TC and OE33 are in RPMI1640, 10% FBS, 2 mM L-glutamine, 50 U / mL penicillin and 50 ⁇ g / Culturing was performed using mL of streptomycin added.
  • IEC-18 was cultured in DMEM supplemented with 5% FBS, 1 U insulin, 2 mM L-glutamine, 50 U / mL penicillin and 50 ⁇ g / mL streptomycin.
  • Mouse primary intestinal epithelial cells were prepared and cultured according to the method described previously (Liu X et al., Am J Pathol. 2012 Feb; 180 (2): 599-607).
  • L. casei ATCC 334, LGG ATCC 53103, L. Coryniformis ATCC 25600 and L. Fermentis ATCC 23271 was purchased from ATCC. These microorganisms were cultured overnight at 37 ° C. using MRS broth (Difco), transferred to MEM, and cultured for one day. The culture medium after centrifugation was centrifuged at 5000 ⁇ g for 10 minutes to recover the supernatant, which was filtered through a 0.2 ⁇ m filter and used as the culture supernatant in the following examples.
  • n 5 unless otherwise specified
  • the measurement sample was subjected to a final concentration of 10 to 10 ⁇ g / Incubation was started by adding to DMEM to make the mL, and the plate was collected after 24, 48, 72 or 96 hours.
  • 5% TCA was added to each well and left at 4 ° C. for 1 hour, followed by washing 4 times with pure water.
  • the plate was dried at room temperature, 100 ⁇ L of 0.057 wt% SRB aqueous solution was added to each well to stain the cells, washed 4 times with 0.1% acetic acid and dried. The cell density at each incubation time was measured by measuring the OD at 510 nm when the stained cells were dissolved in 10 mM Tris buffer.
  • Example 1 1) Antitumor activity of the culture supernatant
  • the colon cancer cell lines Caco2 / bbe, SKCO-1 and SW620 were used as test cells, and LGG ATCC 53103, L. casei ATCC 334, L.C. Coryniformis ATCC 25600 and L.
  • Each culture supernatant of fermentis ATCC23271 was used as a measurement sample, and the antitumor activity of each supernatant was measured by SRB assay. The result is shown in FIG. For any colorectal cancer cell line, L.
  • the culture supernatant of casei ATCC 334 was confirmed to exhibit strong antitumor activity.
  • a fraction with a confirmed antitumor activity was collected by subjecting the fraction having a molecular weight of 0.5 kDa to 3 kDa to gel filtration chromatography using AKTA-HPLC system (GE healthcare) equipped with a superdex peptide column.
  • AKTA-HPLC system GE healthcare
  • the final antitumor activity fraction was treated with protease, but no change was observed in the antitumor activity.
  • amino acid analysis, sugar chain analysis, and peptidoglycan detection were tried, but none of the amino acid, sugar chain, or peptidoglycan was detected. Further, when atomic absorption analysis was attempted, iron, zinc and calcium were detected.
  • FIG. 6 shows the appearance of the mouse on the first day after transplantation and 9 days after the transplantation
  • FIG. 7 shows the volume change of the tumor mass. As shown in FIGS. 6 and 7, ferrichrome significantly inhibited the growth of the tumor mass by the transplanted cells.
  • test cells were colon cancer cell line SW620 and intestinal epithelial cell IEC-18, and ferrichrome, 5-fluorouracil (5-FU) and cisplatin, which are used as anticancer agents, were measured as antitumor samples by SRB assay. Activity was compared. As a result, ferrichrome showed antitumor activity against SW620 over 5-FU and cisplatin (FIG. 8). On the other hand, for IEC-18, 5-FU and cisplatin decreased the cell density particularly when used at high concentrations, but it was confirmed that ferrichrome did not significantly affect the cell density (FIG. 9). .
  • ⁇ Test Example 1> Blood AST (aspartate aminotransferase), ALT (alanine aminotransferase), and serum iron when C57 / BL6 mice (n 5) were orally or intravenously administered 10 ⁇ g or 100 ⁇ g / day of ferrichrome over 7 days was measured, but no significant change was observed compared to the control (FIG. 10).
  • ⁇ Test Example 2 Western blotting using a specific antibody (Cell Signaling) against each of cleaved caspase-3 and PARP is performed on the total protein recovered from a colon cancer cell line SW620 treated with ferrichrome using a mammalian cell extraction kit (BioVision). went. As a result, the addition of ferrichrome increased cleaved caspase-3 and PARP in a dose-dependent manner, and induction of apoptosis was observed (FIG. 11). Induction of apoptosis was also confirmed by TUNEL staining (In Situ Cell Death Detection Kit and TMR red (Roche Diagnostics)). FIG. 12 shows the result of the number of TUNEL staining positive cells of SW620 treated with 0.1 ⁇ g / mL ferrichrome.
  • DDIT3 DNA damage-inductive transcript 3
  • Pancreatic cancer cell lines bxpc3 and suite2, gastric cancer cell lines MKN45 and SH-10-TC, and esophageal cancer cell line OE33 were used as test cells, and the dose dependence of antitumor activity of ferrichrome was tested by SRB assay.
  • Ferrichrome exhibited antitumor activity in a dose-dependent manner for all test cells (FIGS. 16 to 18).
  • Ferrichrome also has antitumor activity against pancreatic cancer cell lines (KP3, KP1N, KP3L, Miapaca), gastric cancer cell lines (MKN7, MKN74) and esophageal cancer cell lines (OE19) other than those described above. It was confirmed to show.
  • FIG. 19 shows photographs of the transplanted sites 1 day and 19 days after the transplantation
  • FIG. 20 shows changes in the tumor volume ratio when the tumor volume 1 day after the transplantation is taken as 1.
  • mice treated with ferrichrome The increase in the tumor volume ratio of mice treated with ferrichrome is suppressed compared to that of the control group administered with PBS alone, and the size of the tumor mass at the transplantation site is suppressed to such an extent that it can be clearly distinguished with the naked eye. It had been.
  • mice administered intraperitoneally with ferrichrome was significantly suppressed compared with that of the control group administered with PBS alone.
  • mice (6 weeks old, sputum) were intraperitoneally administered with azoxymethane (AOM) 10 mg / kg.
  • AOM azoxymethane
  • Mice were fed with dextran sulfate sodium (DSS, manufactured by MP bio), which was raised for 1 week after AOM treatment and diluted to 1.5% with distilled water, by free drinking for 1 week.
  • DSS dextran sulfate sodium
  • a chemical carcinogenesis model was prepared by administering dextran sulfate sodium (DSS, manufactured by MP Bio) diluted to 1% with distilled water to mice with free drinking water for 1 week.
  • ferrichrome diluted in PBS was orally administered every day, and was bred for 28 days in a normal breeding environment. After the breeding, the large intestine was removed from the mouse, and the tumor area was measured.
  • a normal mouse to which AOM and DSS were not administered and a chemical carcinogenic mouse to which only PBS was orally administered were prepared, and the tumor area in the large intestine was measured in the same manner.
  • a photograph showing the state of the large intestine of each mouse is shown in FIG. 23, and the tumor area in the large intestine is shown in FIG.
  • mice (6 weeks old, sputum) were intraperitoneally administered with azoxymethane (AOM) 10 mg / kg.
  • AOM azoxymethane
  • Mice were fed with dextran sulfate sodium (DSS, manufactured by MP bio), which was raised for 1 week after AOM treatment and diluted to 1% with distilled water, by free drinking for 1 week.
  • DSS dextran sulfate sodium
  • 100 ⁇ g of ferrichrome dissolved in PBS was intraperitoneally administered at a frequency of once every 2 days, and was bred for 49 days in a normal breeding environment. After the breeding, the large intestine was removed from the mouse, and the tumor area was measured.
  • a normal mouse to which AOM and DSS were not administered and a chemical carcinogenic mouse to which only PBS was administered intraperitoneally were prepared, and the tumor area in the large intestine was measured in the same manner.
  • a photograph showing the state of the large intestine of each mouse is shown in FIG. 25, and the tumor area in the large intestine is shown in FIG.
  • the antitumor agent of the present invention has industrial applicability in the production of a medicine.

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Abstract

[Problem] To provide a highly safe and efficacious antitumor agent that is derived from probiotics. [Solution] The present invention relates to an antitumor agent comprising a compound represented by formula (1) or a complex thereof as an active ingredient. In formula (1): R1 represents a hydrogen atom or a hydroxymethyl group; R2 represents a hydrogen atom, a methyl group or a hydroxymethyl group; and R3, R4 and R5 each independently represent a methyl group, an N5-(trans-5-hydroxy-3-methylpent-2-enoyl) group, an N5-(cis-5-hydroxy-3-methylpent-2-enoyl) group or an N5-(trans-4-carboxy-3-methylpent-2-enoyl) group. According to the present invention, a highly safe and efficacious antitumor agent can be provided.

Description

抗腫瘍剤Antitumor agent
　本発明は、抗腫瘍剤、特に副作用のおそれが少なく、抗腫瘍効果に優れる抗腫瘍剤及びその利用に関する。 The present invention relates to an antitumor agent, in particular, an antitumor agent having a low risk of side effects and excellent antitumor effect, and use thereof.
　大腸（盲腸、結腸、直腸）に発生する大腸がんは、２０１４年の日本における死亡率の高いがん種として、男性では第３位、女性では第１位に挙げられている。 Colorectal cancer that develops in the large intestine (cecum, colon, rectum) is ranked third in men and first in women as a cancer type with a high mortality rate in Japan in 2014.
　大腸がんの発症原因は未だ明らかにはされていない。しかし、肉食を中心とするいわゆる西洋型の食習慣と大腸がん発症の関連性、さらには腸内環境、特に異常な腸内細菌叢と大腸がんとの関連性等が指摘されている（例えば非特許文献１、２）。一方、いわゆる善玉菌（プロバイオティクス）に分類されるラクトバチルス属（Ｌａｃｔｏｂａｃｉｌｌｕｓ）細菌又はビフィドバクテリア属（Ｂｉｆｉｄｏｂａｃｔｅｒｉｕｍ）細菌のいくつかが、抗腫瘍活性を有する物質を産生することも報告されている（例えば、非特許文献３、４）。 The cause of colorectal cancer has not been clarified yet. However, it has been pointed out that the so-called western-style eating habits, mainly meat, and the onset of colorectal cancer, as well as the intestinal environment, particularly the relationship between abnormal intestinal bacterial flora and colorectal cancer, etc. ( For example, non-patent documents 1, 2). On the other hand, some Lactobacillus bacteria or Bifidobacterium bacteria classified as so-called good bacteria (probiotics) have also been reported to produce substances having antitumor activity. (For example, Non-Patent Documents 3 and 4).
　プロバイオティクスの摂取が有害事象をほとんどもたらすことなく健康維持又は増進に寄与することは、古くから提唱され、また証明されてきている。したがって、プロバイオティクス由来の抗腫瘍活性を有する有用成分を見いだし、これを利用することは、副作用の少ない安全な抗腫瘍剤及びこれを用いたがんの治療法の提供につながるものと期待される。 It has been proposed and proven for a long time that the intake of probiotics contributes to maintaining or improving health with few adverse events. Therefore, it is expected that finding useful components with anti-tumor activity derived from probiotics and using them will provide safe anti-tumor agents with few side effects and therapies for cancer using them. The
　本発明は、安全性かつ有効性の高い抗腫瘍剤を提供することを目的とするものである。 The object of the present invention is to provide a safe and highly effective antitumor agent.
　本発明者らは、ラクトバチルス属細菌の培養上清中に高い抗腫瘍活性を有する物質を見いだし、下記の各発明を完成させた。 The present inventors have found a substance having high antitumor activity in the culture supernatant of Lactobacillus bacteria, and have completed the following inventions.
（１）下記式（１）で示される化合物又はその錯体を有効成分とする抗腫瘍剤。
Figure JPOXMLDOC01-appb-C000002
 　（式中、Ｒ１は水素原子又はヒドロキシメチル基を示し；Ｒ２は水素原子、メチル基又はヒドロキシメチル基を示し；Ｒ３、Ｒ４、Ｒ５は、それぞれ独立して、メチル基、Ｎ５－（トランス－５－ヒドロキシ－３－メチルペント－２－エノイル）基、Ｎ５－（シス－５－ヒドロキシ－３－メチルペント－２－エノイル）基又はＮ５－（トランス－４－カルボキシ－３－メチルペント－２－エノイル）基を示す。）
（２）式中、Ｒ１及びＲ２が水素原子、並びにＲ３～Ｒ５がいずれもメチル基である、（１）に記載の抗腫瘍剤。
（３）腫瘍が消化器がんである、（１）又は（２）に記載の抗腫瘍剤。 (1) An antitumor agent comprising a compound represented by the following formula (1) or a complex thereof as an active ingredient.
Figure JPOXMLDOC01-appb-C000002
 (In the formula, R 1 represents a hydrogen atom or a hydroxymethyl group; R 2 represents a hydrogen atom, a methyl group or a hydroxymethyl group; R 3 , R 4 and R 5 each independently represents a methyl group, N 5 - (trans-5-hydroxy-3-methylpent-2-enoyl) group, N 5 - (cis-5-hydroxy-3-methylpent-2-enoyl) group or N 5 - (trans-4-carboxy -3 -Methylpent-2-enoyl) group.)
(2) The antitumor agent according to (1), wherein R 1 and R 2 are hydrogen atoms, and R 3 to R 5 are all methyl groups.
(3) The antitumor agent according to (1) or (2), wherein the tumor is a digestive cancer.
　本発明によれば、抗腫瘍剤として臨床現場で使用されている５－フルオロウラシル又はシスプラチンと同等以上の抗腫瘍活性を示す一方、肝毒性等の有害事象をもたらすことのない、有効性かつ安全性の高い抗腫瘍剤を提供することが可能になる。 According to the present invention, the anti-tumor activity equivalent to or better than 5-fluorouracil or cisplatin used in clinical practice as an anti-tumor agent, while being effective and safe without causing adverse events such as hepatotoxicity. It is possible to provide a high antitumor agent.
ラクトバチルス属細菌であるＬａｃｔｏｂａｃｉｌｌｕｓ　ＧＧ　ＡＴＣＣ５３１０３、Ｌ．ｃａｓｅｉ　ＡＴＣＣ３３４、Ｌ．ｃｏｒｙｎｉｆｏｒｍｉｓ　ＡＴＣＣ２５６００及びＬ．ｆｅｒｍｅｎｔｉｓ　ＡＴＣＣ２３２７１の各培養上清の、大腸がん細胞株に対する抗腫瘍活性を示すグラフである。パネルＡは大腸がん細胞株Ｃａｃｏ２／ｂｂｅ、パネルＢは大腸がん細胞株ＳＫＣＯ１、パネルＣは大腸がん細胞株ＳＷ６２０に対する抗腫瘍活性をそれぞれ示す。Lactobacillus GG ATCC 53103, a L. lactobacillus bacterium. casei ATCC 334, L.C. Coryniformis ATCC 25600 and L. 2 is a graph showing the antitumor activity of each culture supernatant of fermentis ATCC 23271 against colon cancer cell lines. Panel A shows anti-tumor activity against colon cancer cell line Caco2 / bbe, Panel B shows colon cancer cell line SKCO1, and Panel C shows anti-tumor activity against colon cancer cell line SW620. Ｌ．ｃａｓｅｉ　ＡＴＣＣ３３４の培養上清から精製された抗腫瘍活性を示す化合物のＨＰＬＣクロマトグラフィーのチャートである。L. 2 is a HPLC chromatography chart of a compound showing antitumor activity purified from a culture supernatant of casei ATCC334. パネルＡは精製された抗腫瘍活性を示す画分の、パネルＢは市販フェリクロームの質量分析結果を示すチャートである。Panel A is a fraction showing the purified anti-tumor activity, and Panel B is a chart showing the results of mass spectrometry of commercially available ferrichrome. 大腸がん細胞株Ｃａｃｏ２／ｂｂｅ（パネルＡ）及び大腸がん細胞株ＳＷ６２０（パネルＢ）に対するフェリクロームの抗腫瘍活性の用量依存性を示すグラフである。It is a graph which shows the dose dependence of the antitumor activity of ferrichrome with respect to colon cancer cell line Caco2 / bbe (panel A) and colon cancer cell line SW620 (panel B). ラット腸上皮細胞ＩＥＣ－１８（パネルＡ）及びマウスプライマリー腸上皮細胞（パネルＢ）に対するフェリクロームの影響を示すグラフである。FIG. 6 is a graph showing the effect of ferrichrome on rat intestinal epithelial cells IEC-18 (panel A) and mouse primary intestinal epithelial cells (panel B). 大腸がん細胞株ＳＷ６２０を移植したヌードマウスにフェリクロームを投与したときの腫瘍塊の成長を示す写真である。上は移植後１日、下は移植後９日の写真である。It is a photograph showing the growth of a tumor mass when ferrichrome is administered to a nude mouse transplanted with a colon cancer cell line SW620. The upper photo is one day after transplantation, and the lower photo is nine days after transplantation. 大腸がん細胞株ＳＷ６２０を移植したヌードマウスにフェリクロームを投与したときの腫瘍塊の体積変化を示すグラフである。It is a graph which shows the volume change of the tumor mass when a ferrichrome is administered to the nude mouse which transplanted colon cancer cell strain SW620. フェリクローム（パネルＡ）、５－フルオロウラシル（パネルＢ）及びシスプラチン（パネルＣ）の大腸がん細胞株ＳＷ６２０に対する抗腫瘍効果を比較したグラフである。FIG. 6 is a graph comparing the antitumor effects of ferrichrome (panel A), 5-fluorouracil (panel B) and cisplatin (panel C) on colon cancer cell line SW620. フェリクローム（パネルＡ）、５－フルオロウラシル（パネルＢ）及びシスプラチン（パネルＣ）のラット腸上皮細胞ＩＥＣ－１８に対する影響を比較したグラフである。FIG. 6 is a graph comparing the effects of ferrichrome (panel A), 5-fluorouracil (panel B) and cisplatin (panel C) on rat intestinal epithelial cells IEC-18. マウスにフェリクロームを経口又は静脈投与したときの血中ＡＳＴ（パネルＡ）、ＡＬＴ（パネルＢ）及び血清鉄（パネルＣ）の測定結果を示すグラフである。It is a graph which shows the measurement result of blood AST (panel A), ALT (panel B), and serum iron (panel C) when ferrichrome is orally or intravenously administered to a mouse. フェリクロームで処理した大腸がん細胞株ＳＷ６２０におけるｃｌｅａｖｅｄ　ｃａｓｐａｓｅ－３及びＰＡＲＰの発現をウェスタンブロッティングで測定した結果を示す写真である。It is a photograph showing the results of measuring the expression of cleaved caspase-3 and PARP in colon cancer cell line SW620 treated with ferrichrome by Western blotting. ０．１μｇ／ｍＬのフェリクロームで処理した大腸がん細胞株ＳＷ６２０のＴＵＮＥＬ染色陽性細胞数を示すグラフである。It is a graph which shows the number of TUNEL staining positive cells of colon cancer cell line SW620 treated with 0.1 μg / mL of ferrichrome. フェリクロームで処理した大腸がん細胞株ＳＷ６２０におけるＤＤＩＴ３の発現量の亢進を、ＲＴ－ＰＣＲ（パネルＡ）及びウェスタンブロッティング（パネルＢ）で測定した結果を示す写真である。It is a photograph showing the results of measuring the increase in the expression level of DDIT3 in colon cancer cell line SW620 treated with ferrichrome by RT-PCR (panel A) and Western blotting (panel B). フェリクロームで処理した大腸がん細胞株ＳＷ６２０におけるｐｈｏｓｐｈｏ－Ａｋｔ、ＪＮＫ、ＥＲＫ、ｐ３８ＭＡＰＫ及びＧＳＫ３βの発現をウェスタンブロッティングで測定した結果を示す写真である。It is a photograph showing the results of measuring the expression of phospho-Akt, JNK, ERK, p38MAPK and GSK3β in a colon cancer cell line SW620 treated with ferrichrome by Western blotting. フェリクロームと共にＪＮＫ経路の阻害剤であるＳＰ６００１２５を大腸がん細胞株ＳＷ６２０に添加したときの抗腫瘍活性の変化を示すグラフである。It is a graph which shows the change of anti-tumor activity when SP600125 which is an inhibitor of JNK pathway is added to colon cancer cell line SW620 with ferrichrome. 膵臓がん細胞株ｂｘｐｃ３（パネルＡ）及びｓｕｉｔ２（パネルＢ）に対するフェリクロームの抗腫瘍活性の用量依存性を示すグラフである。縦軸は細胞密度（５１０ｎｍにおけるＯＤ）を、横軸はフェリクローム添加後の培養時間を表す。It is a graph which shows the dose dependence of the antitumor activity of ferrichrome with respect to pancreatic cancer cell lines bxpc3 (panel A) and suite2 (panel B). The vertical axis represents cell density (OD at 510 nm), and the horizontal axis represents culture time after addition of ferrichrome. 胃がん細胞株ＭＫＮ４５（パネルＡ）及びＳＨ－１０－ＴＣ（パネルＢ）に対するフェリクロームの抗腫瘍活性の用量依存性を示すグラフである。縦軸は細胞密度（５１０ｎｍにおけるＯＤ）を、横軸はフェリクローム添加後の培養時間を表す。FIG. 6 is a graph showing the dose dependence of antitumor activity of ferrichrome against gastric cancer cell lines MKN45 (panel A) and SH-10-TC (panel B). The vertical axis represents cell density (OD at 510 nm), and the horizontal axis represents culture time after addition of ferrichrome. 食道がん細胞株ＯＥ３３に対するフェリクロームの抗腫瘍活性の用量依存性を示すグラフである。縦軸は細胞密度（５１０ｎｍにおけるＯＤ）を、横軸はフェリクローム添加後の培養時間を表す。It is a graph which shows the dose dependence of the antitumor activity of ferrichrome with respect to esophageal cancer cell strain OE33. The vertical axis represents cell density (OD at 510 nm), and the horizontal axis represents culture time after addition of ferrichrome. 胃がん細胞株ＭＫＮ４５を移植したヌードマウスにフェリクロームを投与したときの腫瘍塊の成長を示す写真である。It is a photograph which shows the growth of the tumor mass when ferrichrome is administered to the nude mouse which transplanted gastric cancer cell line MKN45. 胃がん細胞株ＭＫＮ４５を移植したヌードマウスにフェリクロームを投与したときの腫瘍体積比の変化を示すグラフである。It is a graph which shows the change of the tumor volume ratio when a ferrichrome is administered to the nude mouse which transplanted gastric cancer cell strain MKN45. 膵臓がん細胞株Ｓｕｉｔ２を移植したヌードマウスにフェリクロームを投与したときの、がん細胞移植後１２日目の腫瘍塊を示す写真である。It is a photograph showing a tumor mass on the 12th day after cancer cell transplantation when ferrichrome is administered to a nude mouse transplanted with pancreatic cancer cell line Suite2. 膵臓がん細胞株Ｓｕｉｔ２を移植したヌードマウスにフェリクロームを投与したときの腫瘍体積の変化を示すグラフである。It is a graph which shows the change of a tumor volume when ferrichrome is administered to the nude mouse which transplanted pancreatic cancer cell line Suite2. ＰＢＳを経口投与した正常マウス（ＰＢＳ）、ＰＢＳを経口投与した化学発癌マウス（ＡＯＭ－ＤＳＳ，ＰＢＳ）、及びフェリクロームを経口投与した化学発癌マウス（ＡＯＭ－ＤＳＳ，ＦＣ）から摘出した大腸の写真である。Photographs of the large intestine excised from normal mice orally administered PBS (PBS), chemically carcinogenic mice orally administered PBS (AOM-DSS, PBS), and chemically carcinogenic mice orally administered ferrichrome (AOM-DSS, FC) It is. ＰＢＳを経口投与した正常マウス（ＰＢＳ）、ＰＢＳを経口投与した化学発癌マウス（ＡＯＭ－ＤＳＳ　ＰＢＳ）、及びフェリクロームを経口投与した化学発癌マウス（ＡＯＭ－ＤＳＳ　ＦＣ）の大腸における腫瘍面積を示すグラフである。Graph showing the tumor area in the large intestine of normal mice (PBS) orally administered PBS, chemically carcinogenic mice (AOM-DSS PBS) orally administered PBS, and chemically carcinogenic mice (AOM-DSS FC) orally administered ferrichrome It is. ＰＢＳを腹腔内投与した正常マウス（ＰＢＳ）、ＰＢＳを腹腔内投与した化学発癌マウス（ＡＯＭ－ＤＳＳ，ＰＢＳ）、及びフェリクロームを腹腔内投与した化学発癌マウス（ＡＯＭ－ＤＳＳ，ＦＣ）から摘出した大腸の写真である。Extracted from normal mice (PBS) intraperitoneally administered with PBS, chemically carcinogenic mice (AOM-DSS, PBS) intraperitoneally administered with PBS, and chemically carcinogenic mice (AOM-DSS, FC) administered intraperitoneally with ferrichrome It is a photograph of the large intestine. ＰＢＳを腹腔内投与した正常マウス（ＰＢＳ）、ＰＢＳを腹腔内投与した化学発癌マウス（ＡＯＭ－ＤＳＳ　ＰＢＳ）、及びフェリクロームを腹腔内投与した化学発癌マウス（ＡＯＭ－ＤＳＳ　ＦＣ）の大腸における腫瘍面積を示すグラフである。Tumor area in the large intestine of normal mice (PBS) administered intraperitoneally with PBS, chemically carcinogenic mice (AOM-DSS PBS) administered intraperitoneally with PBS, and chemically carcinogenic mice (AOM-DSS FC) administered intraperitoneally with ferrichrome It is a graph which shows.
　本発明の第一の態様は、下記式（１）で示される化合物又はその錯体を有効成分とする抗腫瘍剤に関する。
Figure JPOXMLDOC01-appb-C000003
 　式中、Ｒ１は水素原子又はヒドロキシメチル基を示し；Ｒ２は水素原子、メチル基又はヒドロキシメチル基を示し；Ｒ３、Ｒ４、Ｒ５は、それぞれ独立して、メチル基、Ｎ５－（トランス－５－ヒドロキシ－３－メチルペント－２－エノイル）基、Ｎ５－（シス－５－ヒドロキシ－３－メチルペント－２－エノイル）基又はＮ５－（トランス－４－カルボキシ－３－メチルペント－２－エノイル）基を示す。 The 1st aspect of this invention is related with the antitumor agent which uses the compound or its complex shown by following formula (1) as an active ingredient.
Figure JPOXMLDOC01-appb-C000003
 In the formula, R 1 represents a hydrogen atom or a hydroxymethyl group; R 2 represents a hydrogen atom, a methyl group or a hydroxymethyl group; R 3 , R 4 and R 5 each independently represents a methyl group, N 5 - (trans-5-hydroxy-3-methylpent-2-enoyl) group, N 5 - (cis-5-hydroxy-3-methylpent-2-enoyl) group or N 5 - (trans-4-carboxy-3 Methylpent-2-enoyl) group.
　式（１）に示される化合物は、シデロフォア（鉄イオンと安定な錯体を形成する微生物由来のキレート化合物）の一種として、例えば特許文献１（特開２００９－２８０３０号公報）において報告されている環状タンパク質の一種である。式（１）中のＲ１及びＲ２が水素原子、並びにＲ３～Ｒ５がいずれもメチル基である化合物は、一般にフェリクローム（Ｆｅｒｒｉｃｈｒｏｍｅ）と呼ばれている。特許文献１では、式（１）に示される化合物は、糸状菌の一種である麹菌（Ａｓｐｅｒｇｉｌｌｕｓ　ｏｒｙｚａｅ）によって産生されるシデロフォアとして報告されている。 The compound represented by the formula (1) is a kind of siderophore (a chelate compound derived from a microorganism that forms a stable complex with an iron ion), for example, as described in Patent Document 1 (Japanese Patent Laid-Open No. 2009-28030). It is a kind of protein. A compound in which R 1 and R 2 in the formula (1) are hydrogen atoms and R 3 to R 5 are all methyl groups is generally called ferrichrome. In Patent Document 1, the compound represented by the formula (1) is reported as a siderophore produced by Aspergillus oryzae, which is a type of filamentous fungus.
　本発明者らは、Ａｍｅｒｉｃａｎ　Ｔｙｐｅ　Ｃｕｌｔｕｒｅ　Ｃｏｌｌｅｃｔｉｏｎ（ＡＴＣＣ）に登録、保存されているプロバイオティクスの一種であるラクトバチルス　カゼイ（Ｌ．ｃａｓｅｉ）ＡＴＣＣ３３４の培養上清が、大腸がん、膵臓がん、胃がん、食道がんといった様々ながん細胞の増殖に対して強い抗腫瘍活性を示し、さらにかかる活性がフェリクロームによってもたらされることを確認した。これまでに２６０種以上のシデロフォアが報告されているが（ＳｉｄｅｒｏｆｏｒｅＢａｓｅ、ｈｔｔｐ：／／ｂｅｒｔｒａｎｄｓａｍｕｅｌ．ｆｒｅｅ．ｆｒ／ｓｉｄｅｒｏｐｈｏｒｅ＿ｂａｓｅ／ｉｎｄｅｘ．ｐｈｐ）、フェリクロームが抗腫瘍活性を有していることは、新規な知見である。なお、式（１）に示される化合物の金属イオン、特に鉄イオンとの錯体も、本発明において利用される化合物に包含される。 The inventors of the present invention have developed a culture supernatant of L. casei ATCC 334, which is a type of probiotics registered and stored in the American Type Culture Collection (ATCC), for colon cancer, pancreatic cancer, It showed strong antitumor activity against the growth of various cancer cells such as gastric cancer and esophageal cancer, and confirmed that such activity was brought about by ferrichrome. To date, more than 260 types of siderophores have been reported (SideroforeBase, http://bertrandsamuel.free.fr/siderophore_base/index.php), but it is novel that ferrichrome has antitumor activity. It is knowledge. In addition, the metal ion of the compound shown by Formula (1), especially the complex with an iron ion is also included in the compound utilized in this invention.
　式（１）に示される化合物は、前記特許文献１に記載された培養条件でアスペルギルス・オリゼ（Ａｓｐｅｒｇｉｌｌｕｓ　ｏｒｙｚａｅ）３１２９－７株（ＦＥＲＭ　Ｐ－２０９６１）を培養することによって、製造することができる。 The compound represented by the formula (1) can be produced by culturing Aspergillus oryzae strain 3129-7 (FERM P-20961) under the culture conditions described in Patent Document 1.
　また、フェリクロームは、Ｌ．ｃａｓｅｉ、特にＬ．ｃａｓｅｉ　ＡＴＣＣ３３４を適当な培地で培養することによっても製造することができる。好ましくは、Ｌ．ｃａｓｅｉ　ＡＴＣＣ３３４を、ラクトバチルス属細菌の培養に好適な培地例えばＭａｎ－Ｒｏｇｏｓａ－Ｓｈａｒｐｅ（ＭＲＳ）ブロス（Ｄｉｆｃｏ社）、Ｍｉｎｉｍａｌ　Ｅｓｓｅｎｔｉａｌ　Ｍｅｄｉａ（サーモフィッシャーサイエンティフィック社）などの液体培地で培養して得られる培養上清から、ゲル濾過、逆相クロマトグラフィー、イオン交換クロマトグラフィー等を適宜組み合わせて精製することで、製造することができる。さらに、フェリクロームは、Ｉｓｏｗａの記載の方法（Ｂｕｌｌｅｔｉｎ　ｏｆ　ｔｈｅ　Ｃｈｅｍｉｃａｌ　Ｓｏｃｉｅｔｙ　ｏｆ　Ｊａｐａｎ　４７（１），２１５－２２０, １９７４）に従って化学合成することもできる。 In addition, ferrichrome is L. casei, especially L. Casei ATCC334 can also be produced by culturing in an appropriate medium. Preferably, L.M. Casei ATCC334 is obtained by culturing in a liquid medium suitable for culturing Lactobacillus bacteria such as Man-Rogosa-Sharp (MRS) broth (Difco), Minimal Essential Media (Thermo Fisher Scientific) From the culture supernatant, it can be produced by appropriately combining gel filtration, reverse phase chromatography, ion exchange chromatography and the like. Furthermore, ferrichrome can also be chemically synthesized according to the method described by Isova (Bulletin of the Chemical Society of Japan 47 (1), 215-220, 1974).
　式（１）の化合物、特にフェリクロームは、そのまま抗腫瘍剤として利用してもよく、さらに、薬学的に許容される緩衝剤、安定剤、保存剤、賦形剤その他の成分及び／又は他の有効成分を含む医薬組成物の形態で利用してもよい。かかる医薬組成物は、本発明の第二の態様である。薬学的に許容される成分は当業者において周知であり、当業者が通常の実施能力の範囲内で、例えば第十六改正日本薬局方その他の規格書に記載された成分から製剤の形態に応じて適宜選択して使用することができる。 The compound of the formula (1), particularly ferrichrome, may be used as an antitumor agent as it is, and further, a pharmaceutically acceptable buffer, stabilizer, preservative, excipient and other components and / or others. It may be used in the form of a pharmaceutical composition containing the active ingredients. Such a pharmaceutical composition is the second aspect of the present invention. Pharmaceutically acceptable ingredients are well known to those skilled in the art, and those skilled in the art can use the ingredients from the ingredients described in the 16th revised Japanese Pharmacopoeia and other standards within the scope of their normal performance. Can be selected and used as appropriate.
　本発明の抗腫瘍剤を含む医薬組成物の形態は任意であり、注射剤、点滴剤などの非経口製剤であっても、又は任意選択で適当なコーティングを施した経口投与剤であってもよい。非経口製剤に用いることができる担体としては、例えば、生理食塩水や、ブドウ糖、Ｄ－ソルビトールなどを含む等張液といった水性担体が挙げられる。 The form of the pharmaceutical composition containing the antitumor agent of the present invention is arbitrary, and it may be a parenteral preparation such as an injection or infusion, or an oral administration agent optionally coated with an appropriate coating. Good. Examples of carriers that can be used for parenteral preparations include aqueous carriers such as physiological saline, isotonic solutions containing glucose, D-sorbitol and the like.
　本発明の抗腫瘍剤又はこれを含む医薬組成物の投与方法は、特に制限されないが、非経口製剤である場合は、例えば血管内投与（好ましくは静脈内投与）、腹腔内投与、腸管内投与、皮下投与などを挙げることができる。好ましい実施形態の一つにおいて、本発明の治療剤は、静脈内投与又は経口投与により生体に投与される。本発明の抗腫瘍剤又はこれを含む医薬組成物は、腫瘍の治療に有益なその他の医薬と併用して使用してもよい。 The administration method of the antitumor agent of the present invention or the pharmaceutical composition containing the same is not particularly limited. However, in the case of a parenteral preparation, for example, intravascular administration (preferably intravenous administration), intraperitoneal administration, or intestinal administration. And subcutaneous administration. In one preferred embodiment, the therapeutic agent of the present invention is administered to a living body by intravenous administration or oral administration. The antitumor agent of the present invention or a pharmaceutical composition containing the same may be used in combination with other drugs useful for the treatment of tumors.
　本発明の抗腫瘍剤又はこれを含む医薬組成物の投与量は、用法、患者の年齢、疾患の形態、その他の条件などに応じて適宜選択されるが、通常成人に対して体重１ｋｇあたり１０μｇ～２０００μｇ、好ましくは５０μｇ～１０００μｇ、より好ましくは１００μｇ～５００μｇであり、これを１日に１回若しくは複数回に分けて、又は間歇的に投与することができる。 The dose of the antitumor agent of the present invention or the pharmaceutical composition containing the same is appropriately selected according to the usage, the age of the patient, the form of the disease, other conditions, etc., but usually 10 μg per kg body weight for an adult. It is ˜2000 μg, preferably 50 μg to 1000 μg, more preferably 100 μg to 500 μg, and this can be administered once or several times a day or intermittently.
　この様に、本発明の抗腫瘍剤又はこれを含む医薬組成物は、腫瘍、特に消化器がんの予防及び／又は治療に用いることができ、したがって、本発明の第三の態様は、前記式（１）の化合物、特にフェリクロームを用いた腫瘍、特に消化器がんの予防及び／又は治療の方法も提供するものであるということができる。 Thus, the antitumor agent of the present invention or the pharmaceutical composition containing the same can be used for the prevention and / or treatment of tumors, particularly digestive organ cancers. Therefore, the third aspect of the present invention is the above-mentioned It can be said that the present invention also provides a method for the prevention and / or treatment of tumors, particularly gastrointestinal cancers, using a compound of formula (1), particularly ferrichrome.
　以下、非限定的な実施例を示して、本発明をさらに詳細に説明する。 Hereinafter, the present invention will be described in more detail with reference to non-limiting examples.
＜細胞株、微生物及びその培養＞
　大腸がん細胞株であるＣａｃｏ２／ｂｂｅ（ＡＴＣＣ）、ＳＫＣＯ－１（ＡＴＣＣ）及びＳＷ６２０（ＡＴＣＣ）、膵臓がん細胞株であるｂｘｐｃ３（ＡＴＣＣ）及びＳｕｉｔ２（ヒューマンサイエンス資源バンク）、胃がん細胞株であるＭＫＮ４５（国立研究開発法人　医薬基盤・健康・栄養研究所　ＪＣＲＢ細胞バンク）及びＳＨ－１０－ＴＣ（医療細胞資源センター・細胞バンク）、食道がん細胞株であるＯＥ３３（ＤＳファーマ）、並びにラット腸上皮細胞（ｉｎｔｅｓｔｉｎａｌ　ｅｐｉｔｈｅｒｉａｌ　ｃｅｌｌ）株ＩＥＣ－１８（ＡＴＣＣ）を評価に用いた。 <Cell lines, microorganisms and their culture>
In colon cancer cell lines Caco2 / bbe (ATCC), SKCO-1 (ATCC) and SW620 (ATCC), pancreatic cancer cell lines bxpc3 (ATCC) and Suite2 (Human Science Resource Bank), in gastric cancer cell lines MKN45 (National Research Institute for Healthcare and Nutrition, JCRB Cell Bank) and SH-10-TC (Medical Cell Resource Center / Cell Bank), esophageal cancer cell line OE33 (DS Pharma), and rat Intestinal epithelial cell strain IEC-18 (ATCC) was used for evaluation.
　Ｃａｃｏ２／ｂｂｅ、ＳＫＣＯ１及びＳｕｉｔ２はＤＭＥＭに、またＳＷ６２０、ｂｘｐｃ３、ＭＫＮ－４５、ＳＨ－１０－ＴＣ及びＯＥ３３はＲＰＭＩ１６４０に、それぞれ１０％ＦＢＳ、２ｍＭのＬ－グルタミン、５０Ｕ／ｍＬのペニシリン及び５０μｇ／ｍＬのストレプトマイシンを加えたものを使用して培養した。ＩＥＣ－１８は、５％ＦＢＳ、１Ｕのインスリン、２ｍＭのＬ－グルタミン、５０Ｕ／ｍＬのペニシリン及び５０μｇ／ｍＬのストレプトマイシンを加えたＤＭＥＭを用いて培養した。 Caco2 / bbe, SKCO1 and Suite2 are in DMEM, SW620, bxpc3, MKN-45, SH-10-TC and OE33 are in RPMI1640, 10% FBS, 2 mM L-glutamine, 50 U / mL penicillin and 50 μg / Culturing was performed using mL of streptomycin added. IEC-18 was cultured in DMEM supplemented with 5% FBS, 1 U insulin, 2 mM L-glutamine, 50 U / mL penicillin and 50 μg / mL streptomycin.
　マウスプライマリー腸上皮細胞は既報（Ｌｉｕ　Ｘ　ｅｔ　ａｌ．，Ａｍ　Ｊ　Ｐａｔｈｏｌ．　２０１２　Ｆｅｂ；１８０（２）：５９９－６０７）の方法に従って調製、培養した。 Mouse primary intestinal epithelial cells were prepared and cultured according to the method described previously (Liu X et al., Am J Pathol. 2012 Feb; 180 (2): 599-607).
　Ｌ．ｃａｓｅｉ　ＡＴＣＣ３３４、ＬＧＧ　ＡＴＣＣ５３１０３、Ｌ．ｃｏｒｙｎｉｆｏｒｍｉｓ　ＡＴＣＣ２５６００及びＬ．ｆｅｒｍｅｎｔｉｓ　ＡＴＣＣ２３２７１は、いずれもＡＴＣＣから購入した。これらの微生物を、ＭＲＳブロス（Ｄｉｆｃｏ社）を用いて３７℃で一晩培養し、さらにＭＥＭに移して一日間培養した。培養後の培地を５０００×ｇで１０分間遠心分離して上清を回収し、これを０．２μｍのフィルターを通してろ過したものを以下の実施例で培養上清として用いた。 L. casei ATCC 334, LGG ATCC 53103, L. Coryniformis ATCC 25600 and L. Fermentis ATCC 23271 was purchased from ATCC. These microorganisms were cultured overnight at 37 ° C. using MRS broth (Difco), transferred to MEM, and cultured for one day. The culture medium after centrifugation was centrifuged at 5000 × g for 10 minutes to recover the supernatant, which was filtered through a 0.2 μm filter and used as the culture supernatant in the following examples.
＜ＳＲＢアッセイによる抗腫瘍活性の測定＞
　９６穴プレートに１．０×１０４個／ウェルとなるように分注した被験細胞（以下、特に指定がないかぎりｎ＝５）を２４時間培養した後、測定試料を終濃度１０ｎｇ～１０μｇ／ｍＬとなるようにＤＭＥＭに添加してインキュベーションを開始し、２４時間、４８時間、７２時間又は９６時間後にプレートを回収した。培地を除去した後、各ウェルに５％ＴＣＡを添加して４℃で１時間静置し、純水で４回洗浄した。室温でプレートを乾燥させ、０．０５７重量％のＳＲＢ水溶液１００μＬを各ウェルに加えて細胞を染色し、０．１％酢酸で４回洗浄してから乾燥させた。染色された細胞を１０ｍＭのＴｒｉｓ緩衝液に溶解したときの５１０ｎｍにおけるＯＤを測定することで、各インキュベーション時間における細胞密度を測定した。 <Measurement of antitumor activity by SRB assay>
After culturing test cells (hereinafter referred to as n = 5 unless otherwise specified), dispensed at a concentration of 1.0 × 10 4 cells / well in a 96-well plate for 24 hours, the measurement sample was subjected to a final concentration of 10 to 10 μg / Incubation was started by adding to DMEM to make the mL, and the plate was collected after 24, 48, 72 or 96 hours. After removing the medium, 5% TCA was added to each well and left at 4 ° C. for 1 hour, followed by washing 4 times with pure water. The plate was dried at room temperature, 100 μL of 0.057 wt% SRB aqueous solution was added to each well to stain the cells, washed 4 times with 0.1% acetic acid and dried. The cell density at each incubation time was measured by measuring the OD at 510 nm when the stained cells were dissolved in 10 mM Tris buffer.
＜実施例１＞
１）培養上清の抗腫瘍活性
　大腸がん細胞株Ｃａｃｏ２／ｂｂｅ、ＳＫＣＯ－１及びＳＷ６２０を被験細胞とし、ＬＧＧ　ＡＴＣＣ５３１０３、Ｌ．ｃａｓｅｉ　ＡＴＣＣ３３４、Ｌ．ｃｏｒｙｎｉｆｏｒｍｉｓ　ＡＴＣＣ２５６００及びＬ．ｆｅｒｍｅｎｔｉｓ　ＡＴＣＣ２３２７１の各培養上清を測定試料として、各上清の抗腫瘍活性をＳＲＢアッセイにより測定した。その結果を図１に示す。いずれの大腸がん細胞株に対しても、Ｌ．ｃａｓｅｉ　ＡＴＣＣ３３４の培養上清は強い抗腫瘍活性を示すことが確認された。 <Example 1>
1) Antitumor activity of the culture supernatant The colon cancer cell lines Caco2 / bbe, SKCO-1 and SW620 were used as test cells, and LGG ATCC 53103, L. casei ATCC 334, L.C. Coryniformis ATCC 25600 and L. Each culture supernatant of fermentis ATCC23271 was used as a measurement sample, and the antitumor activity of each supernatant was measured by SRB assay. The result is shown in FIG. For any colorectal cancer cell line, L. The culture supernatant of casei ATCC 334 was confirmed to exhibit strong antitumor activity.
２）抗腫瘍活性物質の精製
　Ｌ．ｃａｓｅｉ　ＡＴＣＣ３３４の培養上清から、分子量３ｋＤａのカットオフスピンカラム（ＧＥ　Ｈｅａｌｔｈｃａｒｅ）を用いて分子量３ｋＤａ以下の画分を得、次いでこの画分に対してＭｉｃｒｏ　Ｆｌｏａｔ－Ａ－Ｌｙｚｅｒ　Ｄｉａｌｙｓｉｓ　Ｄｅｖｉｃｅ（Ｓｐｅｃｔｒｕｍ　Ｌａｂｏｒａｔｏｒｉｅｓ）を用いた透析を行うことで、分子量０．５ｋＤａ～３ｋＤａの画分を回収した。 2) Purification of antitumor active substance From the culture supernatant of casei ATCC 334, a fraction having a molecular weight of 3 kDa or less was obtained using a cut-off spin column (GE Healthcare) having a molecular weight of 3 kDa. The fraction having a molecular weight of 0.5 kDa to 3 kDa was recovered by dialysis using
　分子量０．５ｋＤａ～３ｋＤａの画分に対して、Ｓｕｐｅｒｄｅｘ　ｐｅｐｔｉｄｅ　ｃｏｌｕｍｎを備えたＡＫＴＡ－ＨＰＬＣ　ｓｙｓｔｅｍ（ＧＥ　ｈｅａｌｔｈｃａｒｅ）を用いてゲル濾過クロマトグラフィーを行い、抗腫瘍活性が確認されたフラクションを回収した。 A fraction with a confirmed antitumor activity was collected by subjecting the fraction having a molecular weight of 0.5 kDa to 3 kDa to gel filtration chromatography using AKTA-HPLC system (GE healthcare) equipped with a superdex peptide column.
　回収されたフラクションに対して、Ｌ－ｃｏｌｕｍｎ（Ｃｈｅｍｉｃａｌｓ　Ｅｖａｌｕａｔｉｏｎ　ａｎｄ　Ｒｅｓｅａｒｃｈ　Ｉｎｓｔｉｔｕｔｅ）を用いた逆相ＨＰＬＣ（０．１％ギ酸及び０．１％ギ酸／アセトニトリルのリニアグラジエント）を行い、抗腫瘍活性画分を回収した。 The collected fractions were subjected to reverse phase HPLC (linear gradient of 0.1% formic acid and 0.1% formic acid / acetonitrile) using L-column (Chemicals Evaluation and Research Institute) to obtain an antitumor activity fraction. Was recovered.
　さらに、ＨｉＴｒａｐ－ＤＥＡＥ、ＣＭ及びＳＰの各カラム（いずれもＧＥ　Ｈｅａｌｔｈｃａｒｅ）を用いたイオン交換クロマトグラフィーをこの順序で行い、抗腫瘍活性画分を回収した。さらに、ＺＩＣ－ＨＩＬＩＣカラム（Ｍｅｒｃｋ　Ｍｉｌｌｉｐｏｒｅ）を用いた順相クロマトグラフィーを行い、ＨＰＬＣで単一ピーク（図２）を示す化合物を含む抗腫瘍活性画分を回収した。 Further, ion exchange chromatography using each column of HiTrap-DEAE, CM and SP (all GE Healthcare) was performed in this order to collect the antitumor activity fraction. Further, normal phase chromatography using a ZIC-HILIC column (Merck Millipore) was performed, and an antitumor activity fraction containing a compound showing a single peak (FIG. 2) by HPLC was recovered.
　最終的に得られた抗腫瘍活性画分に対してプロテアーゼ処理を行ったが、抗腫瘍活性に変化は認められなかった。また、アミノ酸分析、糖鎖分析、ペプチドグリカン検出をそれぞれ試みたが、アミノ酸、糖鎖及びペプチドグリカンはいずれも検出されなかった。さらに原子吸光分析を試みたところ、鉄、亜鉛及びカルシウムが検出された。 The final antitumor activity fraction was treated with protease, but no change was observed in the antitumor activity. In addition, amino acid analysis, sugar chain analysis, and peptidoglycan detection were tried, but none of the amino acid, sugar chain, or peptidoglycan was detected. Further, when atomic absorption analysis was attempted, iron, zinc and calcium were detected.
　この画分について、Ｎａｎｏ　Ｆｒｏｎｔｉｅｒ　ｅｌＤ　Ｌｉｑｕｉｄ　Ｃｈｒｏｍａｔｏｇｒａｐｙ　Ｍａｓｓ　Ｓｐｅｃｔｒｏｍｅｔｅｒ（Ｈｉｔａｃｈｉ　Ｈｉｇｈ－ｔｅｃｈｎｏｌｏｇｉｅｓ）を用いて質量分析（ＴＯＦ、イオン化法：マイクロＥＳＩ）を行ったところ、図３Ａに示すチャートが得られた。このチャートにおけるｍ／ｚ７６３．２のピークは、市販のフェリクローム（Ｓｉｇｍａ－Ａｌｄｒｉｃｈ）について同様に質量分析を行って得られたチャート（図３Ｂ）のそれとほぼ一致した。また、市販のフェリクロームを測定試料とし、大腸がん細胞株ＳＷ６２０を被験細胞として抗腫瘍活性を測定したところ、強い抗腫瘍活性が確認された。 This fraction was subjected to mass spectrometry (TOF, ionization method: micro ESI) using Nano Frontier elD Liquid Chromatography Mass Spectrometer (Hitachi High-technologies), and the chart shown in Fig. 3A was obtained. The peak at m / z 763.2 in this chart almost coincided with that of the chart (FIG. 3B) obtained by mass spectrometry of commercially available ferrichrome (Sigma-Aldrich). Further, when the antitumor activity was measured using a commercially available ferrichrome as a measurement sample and the colon cancer cell line SW620 as a test cell, strong antitumor activity was confirmed.
　以上から、Ｌ．ｃａｓｅｉ　ＡＴＣＣ３３４の培養上清に含まれる抗腫瘍活性を有する化合物は、フェリクロームであると特定された。 From the above, L. The compound having antitumor activity contained in the culture supernatant of casei ATCC 334 was identified as ferrichrome.
＜実施例２＞
　被験細胞としてＣａｃｏ２／ｂｂｅ及びＳＷ６２０を用い、フェリクロームの抗腫瘍活性の用量依存性をＳＲＢアッセイにより試験した。同時に、ラット腸上皮細胞株ＩＥＣ－１８及びマウスプライマリー腸上皮細胞に対するフェリクロームの影響を調べた。その結果、フェリクロームはＣａｃｏ２／ｂｂｅ及びＳＷ６２０に対して用量依存的に抗腫瘍活性を示した（図４）が、ＩＥＣ－１８及びマウスプライマリー腸上皮細胞に対しては、細胞密度をあまり低下させなかった（図５）。また、フェリクロームは別の大腸がん細胞株ＨＴ２９、ＨＣＴ１１６及びＳＫＣＯ１に対しても、Ｃａｃｏ２／ｂｂｅ及びＳＷ６２０同様に抗腫瘍活性を示すことが確認された。 <Example 2>
Using Caco2 / bbe and SW620 as test cells, the dose dependence of the antitumor activity of ferrichrome was tested by SRB assay. At the same time, the effect of ferrichrome on rat intestinal epithelial cell line IEC-18 and mouse primary intestinal epithelial cells was examined. As a result, ferrichrome showed an antitumor activity in a dose-dependent manner against Caco2 / bbe and SW620 (FIG. 4), but decreased the cell density much against IEC-18 and mouse primary intestinal epithelial cells. None (Figure 5). In addition, it was confirmed that ferrichrome exhibits antitumor activity against other colon cancer cell lines HT29, HCT116 and SKCO1 as well as Caco2 / bbe and SW620.
＜実施例３＞
　２×１０６個の大腸がん細胞株ＳＷ６２０を、ＢＡＬＢ／ｃヌードマウスの背部皮下に注射して移植した。移植の翌日から毎日、１０μｇ／日のフェリクローム（ｎ＝１６）又はＰＢＳ（ｎ＝１６）を移植部位に投与し、各移植部位における腫瘍塊の成長を９日間観察した。移植後１日目及び移植後９日目のマウスの外観を図６に、腫瘍塊の体積変化を図７にそれぞれ示す。図６及び図７に示されるように、フェリクロームは移植細胞による腫瘍塊の成長を顕著に阻害した。 <Example 3>
2 × 10 6 colon cancer cell lines SW620 were implanted subcutaneously in the back of BALB / c nude mice. Every day from the day after transplantation, 10 μg / day of ferrichrome (n = 16) or PBS (n = 16) was administered to the transplant site, and the growth of the tumor mass at each transplant site was observed for 9 days. FIG. 6 shows the appearance of the mouse on the first day after transplantation and 9 days after the transplantation, and FIG. 7 shows the volume change of the tumor mass. As shown in FIGS. 6 and 7, ferrichrome significantly inhibited the growth of the tumor mass by the transplanted cells.
＜実施例４＞
　被験細胞を大腸がん細胞株ＳＷ６２０及び腸上皮細胞ＩＥＣ－１８とし、フェリクローム、抗がん剤として利用されている５－フルオロウラシル（５－ＦＵ）及びシスプラチンを測定試料として、ＳＲＢアッセイにより抗腫瘍活性を比較した。その結果、フェリクロームは５－ＦＵ及びシスプラチンを上回るＳＷ６２０に対する抗腫瘍活性を示した（図８）。一方、ＩＥＣ－１８に対しては、５－ＦＵ及びシスプラチンは特に高濃度で用いた際に細胞密度を低下させたが、フェリクロームは細胞密度に大きく影響しないことが確認された（図９）。 <Example 4>
The test cells were colon cancer cell line SW620 and intestinal epithelial cell IEC-18, and ferrichrome, 5-fluorouracil (5-FU) and cisplatin, which are used as anticancer agents, were measured as antitumor samples by SRB assay. Activity was compared. As a result, ferrichrome showed antitumor activity against SW620 over 5-FU and cisplatin (FIG. 8). On the other hand, for IEC-18, 5-FU and cisplatin decreased the cell density particularly when used at high concentrations, but it was confirmed that ferrichrome did not significantly affect the cell density (FIG. 9). .
＜試験例１＞
　Ｃ５７／ＢＬ６マウス（ｎ＝５）に７日間に亘ってフェリクローム１０μｇ又は１００μｇ／日を経口投与又は静脈投与したときの血中ＡＳＴ（アスパラギン酸アミノトランスフェラーゼ）、ＡＬＴ（アラニンアミノトランスフェラーゼ）及び血清鉄を測定したが、コントロールと比較して有意な変化は観察されなかった（図１０）。 <Test Example 1>
Blood AST (aspartate aminotransferase), ALT (alanine aminotransferase), and serum iron when C57 / BL6 mice (n = 5) were orally or intravenously administered 10 μg or 100 μg / day of ferrichrome over 7 days Was measured, but no significant change was observed compared to the control (FIG. 10).
＜試験例２＞
　フェリクロームで処理した大腸がん細胞株ＳＷ６２０からｍａｍｍａｌｉａｎ　ｃｅｌｌ　ｅｘｔｒａｃｔｉｏｎ　ｋｉｔ（ＢｉｏＶｉｓｉｏｎ）を用いて回収した総タンパク質に対して、ｃｌｅａｖｅｄ　ｃａｓｐａｓｅ－３及びＰＡＲＰそれぞれに対する特異抗体（Ｃｅｌｌ　Ｓｉｇｎａｌｉｎｇ）を用いたウェスタンブロッティングを行った。その結果、フェリクロームの添加によってｃｌｅａｖｅｄ　ｃａｓｐａｓｅ－３及びＰＡＲＰは用量依存的に増加しており、アポトーシスの誘導が認められた（図１１）。アポトーシスの誘導は、ＴＵＮＥＬ染色（Ｉｎ　Ｓｉｔｕ　Ｃｅｌｌ　Ｄｅａｔｈ　Ｄｅｔｅｃｔｉｏｎ　Ｋｉｔ　ａｎｄ　ＴＭＲ　ｒｅｄ（Ｒｏｃｈｅ　Ｄｉａｇｎｏｓｔｉｃ））によっても確認された。図１２に０．１μｇ／ｍＬのフェリクロームで処理したＳＷ６２０のＴＵＮＥＬ染色陽性細胞数の結果を示す。 <Test Example 2>
Western blotting using a specific antibody (Cell Signaling) against each of cleaved caspase-3 and PARP is performed on the total protein recovered from a colon cancer cell line SW620 treated with ferrichrome using a mammalian cell extraction kit (BioVision). went. As a result, the addition of ferrichrome increased cleaved caspase-3 and PARP in a dose-dependent manner, and induction of apoptosis was observed (FIG. 11). Induction of apoptosis was also confirmed by TUNEL staining (In Situ Cell Death Detection Kit and TMR red (Roche Diagnostics)). FIG. 12 shows the result of the number of TUNEL staining positive cells of SW620 treated with 0.1 μg / mL ferrichrome.
　また、フェリクロームで処理した及び処理しなかった大腸がん細胞株ＳＷ６２０の間でのｍＲＮＡ発現量の変化を、ＭｅｔａＣｏｒｅソフトウェアプログラムを用いたハイスループットシーケンス解析を行って測定した。その結果、フェリクローム処理によって小胞体ストレスに関与する遺伝子の発現に変動が見られ、特にＤＮＡ　ｄａｍａｇｅ－ｉｎｄｕｃｉｂｌｅ　ｔｒａｎｓｃｒｉｐｔ　３（ＤＤＩＴ３）の発現量に顕著な変化（発現の亢進）が観察された。ＤＤＩＴ３はＢａｘ－Ｂａｋミトコンドリア透過及びＪＮＫシグナリングに関与することが知られているタンパク質である（Ｔａｂａｓ　Ｉ，　Ｒｏｎ　Ｄ．　Ｎａｔ　Ｃｅｌｌ　Ｂｉｏｌ．　２０１１　Ｍａｒ；１３（３）：１８４－９０）。上記ＤＤＩＴ３の発現の亢進は、ＲＴ－ＰＣＲ及びウェスタンブロッティングでも確認された（図１３）。 Also, changes in mRNA expression level between the colon cancer cell line SW620 treated and not treated with ferrichrome were measured by performing high-throughput sequence analysis using the MetaCore software program. As a result, the expression of genes involved in endoplasmic reticulum stress was changed by ferrichrome treatment, and in particular, a marked change (enhanced expression) was observed in the expression level of DNA damage-inductive transcript 3 (DDIT3). DDIT3 is a protein known to be involved in Bax-Bak mitochondrial permeation and JNK signaling (Tabas I, Ron D. Nat Cell Biol. 2011 Mar; 13 (3): 184-90). The enhanced expression of DDIT3 was also confirmed by RT-PCR and Western blotting (FIG. 13).
　さらに、フェリクロームで処理したＳＷ６２０から回収した総タンパク質に対して、ｐｈｏｓｐｈｏ－Ａｋｔ、ＪＮＫ、ＥＲＫ、ｐ３８ＭＡＰＫ、ＧＳＫ３βそれぞれに対する特異抗体（Ｃｅｌｌ　Ｓｉｇｎａｌｉｎｇ）を用いたウェスタンブロッティングを行った結果、ＳＷ６２０におけるリン酸化ＪＮＫの発現が亢進していることが確認された（図１４）。また、フェリクローム（０．１μｇ／ｍＬ）及びＪＮＫ経路の阻害剤であるＳＰ６００１２５（１又は５μＭ）の両方をＳＷ６２０に添加すると、フェリクロームの抗腫瘍活性の抑制が認められ（図１５）、ｃｌｅａｖｅｄ　ｃａｓｐａｓｅ－３及びＰＡＲＰの発現も確認された。このｃｌｅａｖｅｄ　ｃａｓｐａｓｅ－３及びＰＡＲＰの発現低下は、フェリクロームを添加したＳＷ６２０をＪＮＫに対するｓｉＲＮＡで処理したときも観察された。 Furthermore, the total protein recovered from SW620 treated with ferrichrome was subjected to Western blotting using specific antibodies (Cell Signaling) for phospho-Akt, JNK, ERK, p38MAPK, and GSK3β. As a result, phosphorylation at SW620 was performed. It was confirmed that the expression of JNK was enhanced (FIG. 14). In addition, when both ferrichrome (0.1 μg / mL) and SP600125 (1 or 5 μM), which is an inhibitor of the JNK pathway, were added to SW620, suppression of the antitumor activity of ferrichrome was observed (FIG. 15), and cleaved. The expression of caspase-3 and PARP was also confirmed. This decrease in the expression of cleaved caspase-3 and PARP was also observed when SW620 supplemented with ferrichrome was treated with siRNA against JNK.
　以上の結果から、フェリクローム添加によるＳＷ６２０の細胞密度の低下は、ＪＮＫ－ＤＤＩＴ３が関与するアポトーシス経路が活性化されることによるものであると推察された。 From the above results, it was speculated that the decrease in the cell density of SW620 due to the addition of ferrichrome was due to the activation of the apoptotic pathway involving JNK-DDIT3.
＜実施例５＞
　被験細胞として膵臓がん細胞株ｂｘｐｃ３及びｓｕｉｔ２、胃がん細胞株ＭＫＮ４５及びＳＨ－１０－ＴＣ、並びに食道がん細胞株ＯＥ３３を用い、フェリクロームの抗腫瘍活性の用量依存性をＳＲＢアッセイにより試験した。フェリクロームはいずれの被験細胞に対しても用量依存的に抗腫瘍活性を示した（図１６～１８）。また、フェリクロームは上記以外の膵臓がん細胞株（ＫＰ３、ＫＰ１Ｎ、ＫＰ３Ｌ、Ｍｉａｐａｃａ）、胃がん細胞株（ＭＫＮ７、ＭＫＮ７４）及び食道がん細胞株（ＯＥ１９）に対しても同様に抗腫瘍活性を示すことが確認された。 <Example 5>
Pancreatic cancer cell lines bxpc3 and suite2, gastric cancer cell lines MKN45 and SH-10-TC, and esophageal cancer cell line OE33 were used as test cells, and the dose dependence of antitumor activity of ferrichrome was tested by SRB assay. Ferrichrome exhibited antitumor activity in a dose-dependent manner for all test cells (FIGS. 16 to 18). Ferrichrome also has antitumor activity against pancreatic cancer cell lines (KP3, KP1N, KP3L, Miapaca), gastric cancer cell lines (MKN7, MKN74) and esophageal cancer cell lines (OE19) other than those described above. It was confirmed to show.
＜実施例６＞
　２群のＢＡＬＢ／ｃヌードマウス（ｎ＝６／群）の背部皮下に１×１０６個の胃がん細胞ＭＫＮ４５を注射して移植した。移植の翌日から毎日、１群にはＰＢＳに溶解したフェリクローム１００μｇを、他の１群にはＰＢＳのみをそれぞれ移植部位に投与しながら、通常の飼育環境下で２１日間飼育した。移植１日後と１９日後の移植部位の写真を図１９に、移植１日後の腫瘍体積を１としたときの腫瘍体積比の変化を図２０に示す。 <Example 6>
Two groups of BALB / c nude mice (n = 6 / group) were implanted subcutaneously in the back with 1 × 10 6 gastric cancer cells MKN45. Daily from the day after transplantation, 100 μg of ferrichrome dissolved in PBS was administered to one group and PBS alone was administered to the other site for 21 days in a normal breeding environment. FIG. 19 shows photographs of the transplanted sites 1 day and 19 days after the transplantation, and FIG. 20 shows changes in the tumor volume ratio when the tumor volume 1 day after the transplantation is taken as 1.
　フェリクロームを投与したマウスの腫瘍体積比の増加は、ＰＢＳのみを投与した対照群のそれと比較して抑制され、また移植部位における腫瘍塊の大きさも肉眼で明確に区別することができる程度に抑制されていた。 The increase in the tumor volume ratio of mice treated with ferrichrome is suppressed compared to that of the control group administered with PBS alone, and the size of the tumor mass at the transplantation site is suppressed to such an extent that it can be clearly distinguished with the naked eye. It had been.
＜実施例７＞
　２群のヌードマウス（ｎ＝５／群）に１×１０６個の膵臓がん細胞Ｓｕｉｔ２を皮下移植した２日後から、１群にはＰＢＳに溶解したフェリクローム１００μｇを、他の１群にはＰＢＳのみをそれぞれ２日に１回、腹腔内投与しながら、通常の飼育環境下で１２日間飼育した。移植１２日後の移植部位の写真を図２１に、腫瘍塊の体積変化を図２２に示す。 <Example 7>
Two days after the transplantation of 1 × 10 6 pancreatic cancer cells Suite2 subcutaneously to two groups of nude mice (n = 5 / group), one group received 100 μg of ferrichrome dissolved in PBS, and the other group The mice were reared for 12 days in a normal rearing environment while intraperitoneally administering PBS alone once every two days. A photograph of the transplanted site 12 days after transplantation is shown in FIG. 21, and the volume change of the tumor mass is shown in FIG.
　フェリクロームを腹腔内投与したマウスの腫瘍体積の増加は、ＰＢＳのみを投与した対照群のそれと比較して顕著に抑制された。 The increase in the tumor volume of mice administered intraperitoneally with ferrichrome was significantly suppressed compared with that of the control group administered with PBS alone.
＜実施例８＞
　ＢＡＬＢ/ｃマウス（６週齢、♂）にアゾキシメタン（ＡＯＭ）１０ｍｇ／ｋｇを腹腔内投与した。ＡＯＭ処置後１週間飼育し、蒸留水で１．５％に希釈したデキストラン硫酸ナトリウム（ＤＳＳ、ＭＰ　ｂｉｏ社製）を１週間自由飲水によりマウスに投与した。１週間休薬したのち、蒸留水で１％に希釈したデキストラン硫酸ナトリウム（ＤＳＳ、ＭＰ　ｂｉｏ社製）を１週間自由飲水によりマウスに投与することで、化学発癌モデルを作製した。再度１週間休薬したのち、ＰＢＳに希釈したフェリクローム５０μｇを毎日経口投与しながら通常の飼育環境下で２８日間飼育した。飼育終了後、マウスから大腸を摘出して、腫瘍面積を測定した。 <Example 8>
BALB / c mice (6 weeks old, sputum) were intraperitoneally administered with azoxymethane (AOM) 10 mg / kg. Mice were fed with dextran sulfate sodium (DSS, manufactured by MP bio), which was raised for 1 week after AOM treatment and diluted to 1.5% with distilled water, by free drinking for 1 week. After a 1-week withdrawal, a chemical carcinogenesis model was prepared by administering dextran sulfate sodium (DSS, manufactured by MP Bio) diluted to 1% with distilled water to mice with free drinking water for 1 week. After resting again for 1 week, 50 µg of ferrichrome diluted in PBS was orally administered every day, and was bred for 28 days in a normal breeding environment. After the breeding, the large intestine was removed from the mouse, and the tumor area was measured.
　ＡＯＭ及びＤＳＳを投与しない正常マウス、及びＰＢＳのみを経口投与した化学発癌マウスをそれぞれ用意し、同様にして大腸における腫瘍面積を測定した。各マウスの大腸の様子を示す写真を図２３に、大腸における腫瘍面積を図２４に示す。 A normal mouse to which AOM and DSS were not administered and a chemical carcinogenic mouse to which only PBS was orally administered were prepared, and the tumor area in the large intestine was measured in the same manner. A photograph showing the state of the large intestine of each mouse is shown in FIG. 23, and the tumor area in the large intestine is shown in FIG.
　フェリクロームを経口投与した化学発癌マウス（ｎ＝７）の大腸における腫瘍面積は、ＰＢＳのみを投与した化学発癌マウス（ｎ＝７）のそれと比較して、有意に抑制された。 The tumor area in the large intestine of a chemically carcinogenic mouse (n = 7) administered orally with ferrichrome was significantly suppressed compared with that of a chemically carcinogenic mouse (n = 7) administered with PBS alone.
＜実施例９＞
　ＢＡＬＢ/ｃマウス（６週齢、♂）にアゾキシメタン（ＡＯＭ）１０ｍｇ／ｋｇを腹腔内投与した。ＡＯＭ処置後１週間飼育し、蒸留水で１％に希釈したデキストラン硫酸ナトリウム（ＤＳＳ、ＭＰ　ｂｉｏ社製）を１週間自由飲水によりマウスに投与した。１週間休薬したのち、ＰＢＳに溶解したフェリクローム１００μｇを２日に１回の頻度で腹腔内投与しながら通常の飼育環境下で４９日間飼育した。飼育終了後、マウスから大腸を摘出して、腫瘍面積を測定した。 <Example 9>
BALB / c mice (6 weeks old, sputum) were intraperitoneally administered with azoxymethane (AOM) 10 mg / kg. Mice were fed with dextran sulfate sodium (DSS, manufactured by MP bio), which was raised for 1 week after AOM treatment and diluted to 1% with distilled water, by free drinking for 1 week. After resting for 1 week, 100 μg of ferrichrome dissolved in PBS was intraperitoneally administered at a frequency of once every 2 days, and was bred for 49 days in a normal breeding environment. After the breeding, the large intestine was removed from the mouse, and the tumor area was measured.
　ＡＯＭ及びＤＳＳを投与しない正常マウス、及びＰＢＳのみを腹腔内投与した化学発癌マウスをそれぞれ用意し、同様にして大腸における腫瘍面積を測定した。各マウスの大腸の様子を示す写真を図２５に、大腸における腫瘍面積を図２６に示す。 A normal mouse to which AOM and DSS were not administered and a chemical carcinogenic mouse to which only PBS was administered intraperitoneally were prepared, and the tumor area in the large intestine was measured in the same manner. A photograph showing the state of the large intestine of each mouse is shown in FIG. 25, and the tumor area in the large intestine is shown in FIG.
　フェリクロームを腹腔内投与した化学発癌マウス（ｎ＝７）の大腸における腫瘍面積は、ＰＢＳのみを腹腔内投与した化学発癌マウス（ｎ＝６）のそれと比較して、有意に抑制された。 The tumor area in the large intestine of chemically carcinogenic mice (n = 7) administered ferrichrome intraperitoneally was significantly suppressed compared to that of chemical carcinogenic mice (n = 6) administered intraperitoneally only PBS.
　本発明の抗腫瘍剤は、医薬の製造における産業上の利用可能性を有する。

  The antitumor agent of the present invention has industrial applicability in the production of a medicine.

Claims (3)

  1. 　下記式（１）で示される化合物又はその錯体を有効成分とする抗腫瘍剤。
    Figure JPOXMLDOC01-appb-C000001
     　（式中、Ｒ１は水素原子又はヒドロキシメチル基を示し；Ｒ２は水素原子、メチル基又はヒドロキシメチル基を示し；Ｒ３、Ｒ４、Ｒ５は、それぞれ独立して、メチル基、Ｎ５－（トランス－５－ヒドロキシ－３－メチルペント－２－エノイル）基、Ｎ５－（シス－５－ヒドロキシ－３－メチルペント－２－エノイル）基又はＮ５－（トランス－４－カルボキシ－３－メチルペント－２－エノイル）基を示す。）     The antitumor agent which uses the compound or its complex shown by following formula (1) as an active ingredient.
    Figure JPOXMLDOC01-appb-C000001
     (In the formula, R 1 represents a hydrogen atom or a hydroxymethyl group; R 2 represents a hydrogen atom, a methyl group or a hydroxymethyl group; R 3 , R 4 and R 5 each independently represents a methyl group, N 5 - (trans-5-hydroxy-3-methylpent-2-enoyl) group, N 5 - (cis-5-hydroxy-3-methylpent-2-enoyl) group or N 5 - (trans-4-carboxy -3 -Methylpent-2-enoyl) group.)
  2. 　式中、Ｒ１及びＲ２が水素原子、並びにＲ３～Ｒ５がいずれもメチル基である、請求項１に記載の抗腫瘍剤。 The antitumor agent according to claim 1, wherein R 1 and R 2 are hydrogen atoms, and R 3 to R 5 are all methyl groups.
  3. 　腫瘍が消化器がんである、請求項１又は２に記載の抗腫瘍剤。

          The antitumor agent according to claim 1 or 2, wherein the tumor is digestive cancer.

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